Two-photon Excited Fluorescence Research Articles

Two-Photon Imaging for Non-Invasive Corneal Examination.

Two-photon imaging (TPI) microscopy, namely, two-photon excited fluorescence (TPEF), fluorescence lifetime imaging (FLIM), and second-harmonic generation (SHG) modalities, has emerged in the past years as a powerful tool for the examination of biological tissues. These modalities rely on different contrast mechanisms and are often used simultaneously to provide complementary information on morphology, metabolism, and structural properties of the imaged tissue. The cornea, being a transparent tissue, rich in collagen and with several cellular layers, is well-suited to be imaged by TPI microscopy. In this review, we discuss the physical principles behind TPI as well as its instrumentation. We also provide an overview of the current advances in TPI instrumentation and image analysis. We describe how TPI can be leveraged to retrieve unique information on the cornea and to complement the information provided by current clinical devices. The present state of corneal TPI is outlined. Finally, we discuss the obstacles that must be overcome and offer perspectives and outlooks to make clinical TPI of the human cornea a reality.

Novel V-shaped D-π-A-π-D two-photon absorption compounds with large Stokes shifts: Synthesis, optical properties, selective detection of cysteine, and imaging in living cells

The novel V-shaped donor-π-acceptor-π-donor compounds bearing an aldehyde group (1Pip, 2EtO, and 3TEG) were designed, synthesized, and evaluated as the two-photon excited fluorescence probes for the selective detection of cysteine (Cys). Their structures were fully characterized by 1H NMR, 13C NMR, FT-IR, and HRMS. The optical properties of the target compounds in various solvents were studied in details and the time-dependent density functional theory calculations were performed to shed light on their electronic structures. It is found that all of them exhibit large Stokes shifts (6331-10047 cm−1). 1Pip and 3TEG are capable of detecting Cys with high selectivity, good anti-interference ability, and favorable two-photon absorption performance. The limits of detection were determined to be equal to 0.15 μM for 1Pip and 0.73 μM for 3TEG. The possible recognition mechanism was also validated by 1H NMR and HRMS. One- and two-photon fluorescence imaging experiments indicate that 3TEG shows more satisfactory cellular membrane permeability than 1Pip, and it can successfully visualize the endogenous and exogenous Cys in living cells. These results imply that 3TEG has potential as a two-photon fluorescence probe for detecting Cys in biological samples.

Enhanced two-photon excited fluorescence from green fluorescent proteins by ultrafast fluctuations in intense light pulse

The parametric down-conversion process in optical parametric generators causes bunching of light due to ultrafast intensity fluctuations, which enhances the efficiency of nonlinear interactions between light and matter. However, the bunching effect in a sufficiently intense light pulse light required for biological nonlinear imaging has not yet been investigated. We demonstrate enhanced two-photon excited fluorescence by ultrafast fluctuations in intense pulse using a wavelength-tunable optical parametric generator consisting of a periodically poled lithium niobate crystal pumped by nanosecond pulses at a wavelength of 532 nm and emitting pulses with a peak power of about 1 kW. The emission wavelength is tuned to about 927 nm, which is optimal for two-photon excitation of green fluorescent protein. The effect of bunching by ultrafast intensity fluctuations in the pulse is evaluated by an autocorrelator using a green fluorescent protein solution as a two-photon absorber. We found an about 1.9-fold enhancement compared with the coherent state of light. Using this calibrated optical parametric generator, we perform two-photon imaging of green fluorescent protein in brain tissue within a timescale of seconds. These experimental results using intense pulses demonstrate that the bunching effect by ultrafast fluctuations can enhance nonlinear imaging in biology and medicine.

Application of Ultrashort Lasers in Developmental Biology: A Review

The evolution of laser technologies and the invention of ultrashort laser pulses have resulted in a sharp jump in laser applications in life sciences. Developmental biology is no exception. The unique ability of ultrashort laser pulses to deposit energy into a microscopic volume in the bulk of transparent material without disrupting the surrounding tissues makes ultrashort lasers a versatile tool for precise microsurgery of cells and subcellular components within structurally complex and fragile specimens like embryos as well as for high-resolution imaging of embryonic processes and developmental mechanisms. Here, we present an overview of recent applications of ultrashort lasers in developmental biology, including techniques of noncontact laser-assisted microsurgery of preimplantation mammalian embryos for oocyte/blastomere enucleation and embryonic cell fusion, as well as techniques of optical transfection and injection for targeted delivery of biomolecules into living embryos and laser-mediated microsurgery of externally developing embryos. Possible applications of ultrashort laser pulses for use in Assisted Reproductive Technologies are also highlighted. Moreover, we discuss various nonlinear optical microscopy techniques (two-photon excited fluorescence, second and third harmonic generation, and coherent Raman scattering) and their application for label-free non-invasive imaging of embryos in their unperturbed state or post-laser-induced modifications.

Temporally shaped vortex phase laser pulses for two-photon excited fluorescence.

We report temporally shaped vortex phase laser pulses for two-photon excited fluorescence of dyes. The particularly tailored pulses are generated by first utilizing a temporal pulse shaper and subsequently a two-dimensional spatial pulse shaper. Various vortex phase shaped structures are demonstrated by combining different two-dimensional phase patterns. Moreover, perpendicular polarization components are used to achieve an enhanced radial two-photon excited fluorescence contrast by applying third order phase functions on the temporal pulse shaper. Particularly, the spatial fluorescence structure is modulated with a combination of Gaussian and vortex phase shaped pulses by modifying only the phase on the temporal modulator. Thereby, interference structures with high spatial resolution arise. The introduced method to generate temporally shaped vortex phase tailored pulses will provide new perspectives for biophotonic applications.

Label-free multimodal imaging of infected Galleria mellonella larvae

Non-linear imaging modalities have enabled us to obtain unique morpho-chemical insights into the tissue architecture of various biological model organisms in a label-free manner. However, these imaging techniques have so far not been applied to analyze the Galleria mellonella infection model. This study utilizes for the first time the strength of multimodal imaging techniques to explore infection-related changes in the Galleria mellonella larvae due to massive E. faecalis bacterial infection. Multimodal imaging techniques such as fluorescent lifetime imaging (FLIM), coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence (TPEF), and second harmonic generation (SHG) were implemented in conjunction with histological HE images to analyze infection-associated tissue damage. The changes in the larvae in response to the infection, such as melanization, vacuolization, nodule formation, and hemocyte infiltration as a defense mechanism of insects against microbial pathogens, were visualized after Enterococcus faecalis was administered. Furthermore, multimodal imaging served for the analysis of implant-associated biofilm infections by visualizing biofilm adherence on medical stainless steel and ePTFE implants within the larvae. Our results suggest that infection-related changes as well as the integrity of the tissue of G. mellonella larvae can be studied with high morphological and chemical contrast in a label-free manner.

Biomimetic design of bioartificial scaffolds for the in vitro modelling of human cardiac fibrosis.

In vitro models of pathological cardiac tissue have attracted interest as predictive platforms for preclinical validation of therapies. However, models reproducing specific pathological features, such as cardiac fibrosis size (i.e., thickness and width) and stage of development are missing. This research was aimed at engineering 2D and 3D models of early-stage post-infarct fibrotic tissue (i.e., characterized by non-aligned tissue organization) on bioartificial scaffolds with biomimetic composition, design, and surface stiffness. 2D scaffolds with random nanofibrous structure and 3D scaffolds with 150µm square-meshed architecture were fabricated from polycaprolactone, surface-grafted with gelatin by mussel-inspired approach and coated with cardiac extracellular matrix (ECM) by 3weeks culture of human cardiac fibroblasts. Scaffold physicochemical properties were thoroughly investigated. AFM analysis of scaffolds in wet state, before cell culture, confirmed their close surface stiffness to human cardiac fibrotic tissue. Following 3weeks culture, biomimetic biophysical and biochemical scaffold properties triggered the activation of myofibroblast phenotype. Upon decellularization, immunostaining, SEM and two-photon excitation fluorescence microscopy showed homogeneous decoration of both 2D and 3D scaffolds with cardiac ECM. The versatility of the approach was demonstrated by culturing ventricular or atrial cardiac fibroblasts on scaffolds, thus suggesting the possibility to use the same scaffold platforms to model both ventricular and atrial cardiac fibrosis. In the future, herein developed in vitro models of cardiac fibrotic tissue, reproducing specific pathological features, will be exploited for a fine preclinical tuning of therapies.

Label-free multimodal nonlinear optical microscopy reveals features of bone composition in pathophysiological conditions.

Bone tissue features a complex microarchitecture and biomolecular composition, which determine biomechanical properties. In addition to state-of-the-art technologies, innovative optical approaches allowing the characterization of the bone in native, label-free conditions can provide new, multi-level insight into this inherently challenging tissue. Here, we exploited multimodal nonlinear optical (NLO) microscopy, including co-registered stimulated Raman scattering, two-photon excited fluorescence, and second-harmonic generation, to image entire vertebrae of murine spine sections. The quantitative nature of these nonlinear interactions allowed us to extract accurate biochemical, morphological, and topological information on the bone tissue and to highlight differences between normal and pathologic samples. Indeed, in a murine model showing bone loss, we observed increased collagen and lipid content as compared to the wild type, along with a decreased craniocaudal alignment of bone collagen fibres. We propose that NLO microscopy can be implemented in standard histopathological analysis of bone in preclinical studies, with the ambitious future perspective to introduce this technique in the clinical practice for the analysis of larger tissue sections.

Long-Term Repeatable In Vivo Monitoring of Amyloid-β Plaques and Vessels in Alzheimer's Disease Mouse Model with Combined TPEF/CARS Microscopy.

Long-term, repeatable monitoring of the appearance and progress of Alzheimer's disease (AD) in real time can be extremely beneficial to acquire highly reliable diagnostic insights, which is crucial for devising apt strategies towards effective AD treatment. Herein, we present an optimized innovative cranial window imaging method for the long-term repeatable imaging of amyloid-β (Aβ) plaques and vessels in an AD mouse model. Basically, two-photon excitation fluorescence (TPEF) microscopy was used to monitor the fluorescently labeled Aβ plaques, whereas the label-free blood vessels were studied using coherent anti-Stokes Raman scattering (CARS) microscopy in the live in vivo AD mouse model. It was possible to clearly observe the Aβ deposition and vascular structure in the target cortex localization for 31 weeks in the AD mouse model using this method. The combined TPEF/CARS imaging studies were also instrumental in realizing the relationship between the tendency of Aβ deposition and ageing. Essentially, the progression of cerebral amyloid angiopathy (CAA) in the AD mouse model was quantitatively characterized, which revealed that the proportion Aβ deposition in the unit vessel can increase from 13.63% to 28.80% upon increasing the age of mice from 8 months old to 14 months old. The proposed imaging method provided an efficient, safe, repeatable platform with simple target localization aptitude towards monitoring the brain tissues, which is an integral part of studying any brain-related physiological or disease conditions to extract crucial structural and functional information.

SURG-24. QUANTITATIVE ANALYSIS OF TUMOR CELL DENSITY AND PROTOPORPHYRIN IX FLOURESCENCE IN GLIOMA PATIENTS USING PAIRED STIMULATED RAMAN HISTOLOGY AND TWO-PHOTON EXCITATION FLUORESCENCE MICROSCOPY

Abstract Fluorescence guidance is widely utilized to improve the precision of cancer surgery. 5-aminolevulinic acid, the most widely used fluorophore in glioma surgery, is thought to cause selective accumulation of fluorescent protoporphyrin IX (PpIX) in tumor cells by exploiting pathologic alterations in heme biosynthetic pathways. PpIX-induced fluorescence is highly specific for densely tumor-infiltrated tissue but less effective for visualizing the tumor periphery. To improve clinical detection of PpIX, we developed a microscope to perform paired stimulated Raman histology and two-photon excitation fluorescence microscopy (TPEF) and validated it in 175 fresh, unprocessed core tumor specimens from 75 high-grade glioma patients and three central nervous system lymphoma patients across three institutions. Surprisingly, intracellular PpIX accumulation was observed primarily in cells with histiocytic, rather than neoplastic morphology, and the number of cells concentrating PpIX within the cytoplasm was associated with the abundance of CD163 positive cells (p< 0.02). There was no correlation between the degree of tumor cellularity and the concentration of PpIX across all imaged specimens (R=-0.21). Our findings encourage reconsideration of the existing theory of 5-ALA-induced tumor cell fluorescence in gliomas and demonstrate how 5-ALA and TPEF imaging can provide a window into the immune microenvironment of human gliomas.

Two-Photon Excitation Spectra of Various Fluorescent Proteins within a Broad Excitation Range.

Two-photon excitation fluorescence laser-scanning microscopy is the preferred method for studying dynamic processes in living organ models or even in living organisms. Thanks to near-infrared and infrared excitation, it is possible to penetrate deep into the tissue, reaching areas of interest relevant to life sciences and biomedicine. In those imaging experiments, two-photon excitation spectra are needed to select the optimal laser wavelength to excite as many fluorophores as possible simultaneously in the sample under consideration. The more fluorophores that can be excited, and the more cell populations that can be studied, the better access to their arrangement and interaction can be reached in complex systems such as immunological organs. However, for many fluorophores, the two-photon excitation properties are poorly predicted from the single-photon spectra and are not yet available, in the literature or databases. Here, we present the broad excitation range (760 nm to 1300 nm) of photon-flux-normalized two-photon spectra of several fluorescent proteins in their cellular environment. This includes the following fluorescent proteins spanning from the cyan to the infrared part of the spectrum: mCerulean3, mTurquoise2, mT-Sapphire, Clover, mKusabiraOrange2, mOrange2, LSS-mOrange, mRuby2, mBeRFP, mCardinal, iRFP670, NirFP, and iRFP720.

Optical properties, bioimaging and theoretical calculation of a Zn(II) complex based on triphenylamine derivative

A luminescent material with various optical properties based on a triphenylamine Zn(II) complex is described. The ultraviolet–visible absorption, one-photon excited fluorescence (OPEF) and two-photon excited fluorescence (TPEF) of the complex indicate that the material has good OPEF and TPEF properties. And the results of one- and two-photon HepG2 cells imaging experiments show the potential of the complex in fluorescence microscopy bioimaging. The experimental Stokes shift and the FWHM (full-width at half-maximum) in different solvents were correlated with the rMPI polarity of the solvent, and the perfect Boltzmann curves were obtained, where the Boltzmann correlation between Stokes shift and solvent polarity is reported for the second time. But the Boltzmann correlation between FWHM and solvent polarity is reported for the first time. In addition, the computational results indicate that, the covalent bond within the salt ZnBr2 is strengthened by the coordination, and the newly formed coordination bond Zn-N is stronger than the original covalent bond Zn-Br.

An exciplex-type thermally activated delayed fluorescence material with wide up-conversion fluorescence excitation window

Exciplexes play an important role in the field of light-emitting materials due to the effective enhancement on fluorescence efficiency through thermally activated delayed fluorescence (TADF) mechanism. This mechanism is also expected to contribute to nonlinear optical applications. Here, we report an exciplex-type TADF material with significant two-photon excited fluorescence (TPEF) properties. It consists of Di-[4-(N,N-di-p-tolyl-amino)-phenyl]cyclohexane (TAPC) as the donor and 1,3,5-Tris(1-phenyl-1H-benzimidazol-2-yl)benzene (TPBi) as the acceptor. The TAPC:TPBi film emits blue fluorescence (peaked at ∼470 nm) under near-infrared excitation. A very wide long-wavelength excitation window of at least 200 nm (660 nm–860 nm) was observed. The reported exciplex material may have the potential to play an important role in the field of one- and two-photon excited fluorescence.

Persistent and Efficient Multimodal Imaging for Tyrosinase Based on Two-Photon Excited Fluorescent and Room-Temperature Phosphorescent Probes.

Tyrosinase is crucial to regulate the metabolism of phenol derivatives, playing an important role in the biosynthesis of melanin pigments, whereas an abnormal level of tyrosinase would lead to severe diseases. It is rather necessary to develop a sensitive and selective imaging tool to assess the level of tyrosinase in vivo. We thoroughly researched the luminous mechanism of the existing TPTYR probe and provided design strategies to improve its two-photon excited fluorescence properties. The designed probes benza2-TPTYR and product benza2-TPTYR-coumarin have large two-photon absorption cross sections at the NIR spectral region (41 GM/706 nm, 71 GM/852 nm), while benza2-TPTYR-coumarin possesses easily distinguishable spectrum in the visible region and a high fluorescence efficiency (ΦF = 0.27). What is more, novel two-photon excited multimodal imaging based on the pure organic small molecule benza1-TPTYR-coumarin (61 GM/936 nm) is proposed first, simultaneously possessing strong instantaneous fluorescent (563.79 nm) and persistent room-temperature phosphorescent emissions (767.68 nm, 0.54 ms).

Excellent ultrafast broadband optical limiting of functionalized twistacenes based on two-photon absorption-induced excited state absorption

Twistacene is an outstanding building block due to the large conjugated system. Herein, a series of twistacenes have been designed and synthesized with the assistant from a theoretical analysis based on the morphology of the natural transition orbitals of first excited state. These twistacenes exhibit two photon absorption and unique broadband excited singlet state absorption features, which is systematically confirmed by transient absorption spectra, two-photon excited fluorescence and z-scan experiments. The overlap of two-photon absorption band with excited state absorption band can improve the OL performance. The experimental results are consistent with theoretical prediction, which confirm the rationalization of molecular design strategy. The screened twistacene 5 exhibits excellent optical limiting behavior with extremely high transmittance and low threshold, where the threshold is 1.3–3.1 mJ/cm2 in a wide optical window from 532 nm to 900 nm in femtosecond regime and 69 mJ/cm2 in picosecond regime. The experimental results suggest twistacene is a promising candidate for nonlinear optical applications, especially for protection against ultrashort laser pulses.

Label-free imaging of M1 and M2 macrophage phenotypes in the human dermis in vivo using two-photon excited FLIM.

Macrophages (ΜΦs) are important immune effector cells that promote (M1 ΜΦs) or inhibit (M2 ΜΦs) inflammation and are involved in numerous physiological and pathogenic immune responses. Their precise role and relevance, however, are not fully understood for lack of noninvasive quantification methods. Here, we show that two-photon excited fluorescence lifetime imaging (TPE-FLIM), a label-free noninvasive method, can visualize ΜΦs in the human dermis in vivo. We demonstrate in vitro that human dermal ΜΦs exhibit specific TPE-FLIM properties that distinguish them from the main components of the extracellular matrix and other dermal cells. We visualized ΜΦs, their phenotypes and phagocytosis in the skin of healthy individuals in vivo using TPE-FLIM. Additionally, machine learning identified M1 and M2 MФs with a sensitivity of 0.88±0.04 and 0.82±0.03 and a specificity of 0.89±0.03 and 0.90±0.03, respectively. In clinical research, TPE-FLIM can advance the understanding of the role of MФs in health and disease.

Minimizing Motion Artifacts in Intravital Microscopy Using the Sedative Effect of Dexmedetomidine.

Among intravital imaging instruments, the intravital two-photon fluorescence excitation microscope has the advantage of enabling real-time 3D fluorescence imaging deep into cells and tissues, with reduced photobleaching and photodamage compared with conventional intravital confocal microscopes. However, excessive motion of organs due to involuntary movement such as breathing may result in out-of-focus images and severe fluorescence intensity fluctuations, which hinder meaningful imaging and analysis. The clinically approved alpha-2 adrenergic receptor agonist dexmedetomidine was administered to mice during two-photon fluorescence intravital imaging to alleviate this problem. As dexmedetomidine blocks the release of the neurotransmitter norepinephrine, pain is suppressed, blood pressure is reduced, and a sedation effect is observed. By tracking the quality of focus and stability of detected fluorescence in two-photon fluorescence images of fluorescein isothiocyanate-sensitized liver vasculature in vivo, we demonstrated that intravascular dexmedetomidine can reduce fluorescence fluctuations caused by respiration on a timescale of minutes in mice, improving image quality and resolution. The results indicate that short-term dexmedetomidine treatment is suitable for reducing involuntary motion in preclinical intravital imaging studies. This method may be applicable to other animal models.

Label-free ex vivo pathological studying on breast cancer tissue by using nonlinear multiphoton and micro-Raman spectral imaging

Raman spectral and multiphoton microscopy (MPM) were utilized in this study to understand the compositional and structural characteristics of the breast tumor microenvironment. Combining these techniques allowed us to address the pathological features of ex vivo healthy and invasive ductal carcinoma (IDC) breast tissue sections from different perspectives. MPM-based two-photon excited fluorescence and second-harmonic generation offer significant advantages for large-area structural tissue imaging. Raman spectral imaging identified the compositional information from a spectroscopic perspective and visualized the specific distribution patterns of biomedical components using a multivariate imaging method (K-mean cluster analysis, KCA). The MPM results revealed the loss of collagen bundles, malignant cell invasion, basement membrane damage, and blood vessel growth in IDC. Additionally, variations in tryptophan, nucleic acid, carotenoid, protein, and lipid content were found in different tissues. The reconstructed Raman images illustrate the morphological alterations of these contents and the edge contours of the cancerous tissue. The acquired multimodal optical results, which extracted complementary information of a sample based on different contrast mechanisms, provided new label-free pathological interpretations of the tumor microenvironment and offered the experimental foundations for developing multimode optical modalities as an immediate intraoperative diagnosis method.

Label-free second harmonic generation imaging of cerebral vascular wall in local ischemia mouse model in vivo

Second harmonic generation (SHG) imaging is label-free and non-invasive, and it has been extensively applied in multiple biological and medical studies, but not in the brain in vivo. In this study, we modified classical two photon excited fluorescence (TPEF) system to perform in vivo simultaneous TPEF and SHG imaging in the local ischemia mouse model. In cerebral vascular walls, we found strong SHG signal, which co-localized with collagen. In the continuous 2 days’ in vivo imaging, this SHG signal remained stable in the local ischemic blood vessel in the initial 4 h, then its signal abruptly increased and got spatially thickened 5 h after thrombosis, and this tendency continued in the following 48 h. This study provides direct and precise timeline of rapid collagen change in cerebral vascular walls in vivo, and reveals the subtle but significant temporal-spatial dynamics of this structural signal during local ischemia. Thus, this cerebral in vivo SHG imaging provides a powerful tool to identify the early and subtle pathological change of collagen around clinical key therapeutic time window.

Quantitative analysis of collagen morphology in breast cancer from millimeter scale using multiphoton microscopy

The tumor microenvironment (TME) is now recognized as an important participant of tumor progression. As the most abundant extracellular matrix component in TME, collagen plays an important role in tumor development. The imaging study of collagen morphological feature in TME is of great significance for understanding the state of tumor. Multiphoton microscopy (MPM), based on second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF), can be used to monitor the morphological changes of biological tissues without labeling. In this study, we used MPM for large-scale imaging of early invasive breast cancer from the tumor center to normal tissues far from the tumor. We found that there were significant differences in collagen morphology between breast cancer tumor boundary, near tumor transition region and normal tissues far from the tumor. Furthermore, the morphological feature of eight collagen fibers was extracted to quantify the variation trend of collagen in three regions. These results may provide a new perspective for the optimal negative margin width of breast-conserving surgery and the understanding of tumor metastasis.